This invention relates generally to the field of bacterial antigens and their use, for example, as immunogenic agents in humans and animals to stimulate an immune response. More specifically, it relates to the vaccination of mammalian species with a polypeptide comprising an alpha helix-forming polypeptide obtained from a choline binding polypeptide as a mechanism for stimulating production of antibodies that protect the vaccine recipient against infection by pathogenic bacterial species. Further, the invention relates to antibodies and antagonists against such polypeptides useful in diagnosis and passive immune therapy with respect to diagnosing and treating such pneumococcal infections.
In a particular aspect, the present invention relates to the prevention and treatment of pneumonococcal infections such as infections of the middle ear, nasopharynx, lung and bronchial areas, blood, CSF, and the like, that are caused by pneumonococcal bacteria. In this regard, certain types of Streptococcus pneumoniae are of particular interest.
S. pneumoniae is a gram positive bacteria which is a major causative agent in invasive infections in animals and humans, such as sepsis, meningitis, otitis media and lobar pneumonia (Tuomanen, et al. NEJM 322:1280-1284 (1995)). As part of the infective process, pneumococci readily bind to non-inflamed human epithelial cells of the upper and lower respiratory tract by binding to eukaryotic carbohydrates in a lectin-like manner (Cundell et al., Micro. Path. 17:361-374 (1994)). Conversion to invasive pneumococcal infections for bound bacteria may involve the local generation of inflammatory factors which may activate the epithelial cells to change the number and type of receptors on their surface (Cundell, et al., Nature, 377:435-438 (1995)). Apparently, one such receptor, platelet activating factor (PAF) is engaged by the pneumococcal bacteria and within a very short period of time (minutes) from the appearance of PAF, pneumococci exhibit strongly enhanced adherence and invasion of tissue. Certain soluble receptor analogs have been shown to prevent the progression of pneumococcal infections (Idanpaan-Heikkila et al., J. Inf. Dis., 176:704-712 (1997)).
A family of choline binding proteins (CBPs), which are non-covalently bound to phosphorylcholine, are present on the surface of pneumococci and have a non-covalent association with teichoic acid or lipoteichoic acid. An example of such family is choline binding protein A (CbpA), an approximately 75 kD weight type of CBP which includes a unique N-terminal domain, a proline rich region, and a C-terminal domain comprised of multiple 20 amino acid repeats responsible for binding to choline. A segment of the N-terminal portion of CbpA protein forms an alpha helix as part of its three-dimensional structure.
Accordingly, it is an object of the present invention to provide a polypeptide having broad protection against pneumococcal infections.
In order to facilitate understanding of the description below and the examples which follow certain frequently occurring methods and/or terms will be described.
xe2x80x9cPlasmidsxe2x80x9d are designated by a lower case p preceded and/or followed by capital letters and/or numbers. The starting plasmids herein are either commercially available, publicly available on an unrestricted basis, or can be constructed from available plasmids in accord with published procedures. In addition, equivalent plasmids to those described are known in the art and will be apparent to the ordinarily skilled artisan.
xe2x80x9cDigestionxe2x80x9d of DNA refers to catalytic cleavage of the DNA with a restriction enzyme that acts only at certain sequences in the DNA. The various restriction enzymes used herein are commercially available and their reaction conditions, cofactors and other requirements were used as would be known to the ordinarily skilled artisan. For analytical purposes, typically 1 xcexcg of plasmid or DNA fragment is used with about 2 units of enzyme in about 20 xcexcl of buffer solution. For the purpose of isolating DNA fragments for plasmid construction, typically 5 to 50 xcexcg of DNA are digested with 20 to 250 units of enzyme in a larger volume. Appropriate buffers and substrate amounts for particular restriction enzymes are specified by the manufacturer. Incubation times of about 1 hour at 37xc2x0 C. are ordinarily used, but may vary in accordance with the supplier""s instructions. After digestion the reaction is electrophoresed directly on a polyacrylamide gel to isolate the desired fragment.
Size separation of the cleaved fragments is performed using 8 percent polyacrylamide gel described by Goeddel, D. et al., Nucleic Acids Res., 8:4057 (1980).
xe2x80x9cOligonucleotidesxe2x80x9d refers to either a single stranded polydeoxynucleotide or two complementary polydeoxynucleotide strands which may be chemically synthesized. Such synthetic oligonucleotides have no 5xe2x80x2 phosphate and thus will not ligate to another oligonucleotide without adding a phosphate with an ATP in the presence of a kinase. A synthetic oligonucleotide will ligate to a fragment that has not been dephosphorylated.
xe2x80x9cLigationxe2x80x9d refers to the process of forming phosphodiester bonds between two double stranded nucleic acid fragments (Maniatis, T., et al., Id., p. 146). Unless otherwise provided, ligation may be accomplished using known buffers and conditions with 10 units to T4 DNA ligase (xe2x80x9cligasexe2x80x9d) per 0.5 xcexcg of approximately equimolar amounts of the DNA fragments to be ligated.
xe2x80x9cHPS portionxe2x80x9d as used herein refers to an amino acid sequence as set forth in SEQ ID NO:2 for a choline binding protein (xe2x80x9cCBPxe2x80x9d) of a pneumococcal bacteria that may be located amino terminal with respect to the proline rich portion of the overall amino acid sequence for such CBP.
The terms xe2x80x9cidentityxe2x80x9d, xe2x80x9c% identityxe2x80x9d or xe2x80x9cpercent identityxe2x80x9d as utilized in this application refer to a calculation of differences between two contiguous sequences which have been aligned for xe2x80x9cbest fitxe2x80x9d (to provide the largest number of aligned identical corresponding sequence elements, wherein elements are either nucleotides or amino acids) and all individual differences are considered as individual difference with respect to the identity. In this respect, all individual element gaps (caused by insertions and deletions with respect to an initial sequence (xe2x80x9creference sequencexe2x80x9d)) over the length of the reference sequence and individual substitutions of different elements (for individual elements of the reference sequence) are considered as individual differences in calculating the total number of differences between two sequences. Individual differences may be compared between two sequences where an initial sequences (reference sequence) has been varied to obtain a variant sequence (comparative sequence) or where a new sequence (comparative sequence) is simply aligned and compared to such a reference sequence. When two aligned sequences are compared all of the individual gaps in BOTH sequences that are caused by the xe2x80x9cbest fitxe2x80x9d alignment over the length of the reference sequence are considered individual differences for the purposes of identity. If an alignment exists which satisfies the stated minimum identity, then a sequence has the stated minimum identity to the reference sequence. For example, the following is a hypothetical comparison of two sequences having 100 elements each that are aligned for best fit wherein one sequence is regarded as the xe2x80x9creference sequencexe2x80x9d and the other as the comparative sequence. All of the individual alignment gaps in both sequences are counted over the length of the reference sequence and added to the number of individual element substitution changes (aligned elements that are different) of the comparative sequence for the total number of element differences. The total number of differences (for example 7 gaps and 3 substitutions) is divided by the total number of elements in the length of the reference sequence (100 elements) for the xe2x80x9cpercentage differencexe2x80x9d (10/100). The resulting percentage difference (10%) is subtracted from 100% identity to provide a xe2x80x9c% identityxe2x80x9d of 90% identity. For the identity calculation all individual differences in both sequences are considered in the above manner over a discrete comparison length (the length of the reference sequence) of two best fit aligned sequences to determine identity. Thus, no algorithm is necessary for such an identity calculation.
xe2x80x9cIsolatedxe2x80x9d in the context of the present invention with respect to polypeptides and/or polynucleotides means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring). For example, a naturally-occurring polynucleotide or polypeptide present in a living organism is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the co-existing materials in the natural system, is isolated. Such polynucleotides could be part of a vector and/or such polynucleotides or polypeptides could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment. The polypeptides and polynucleotides of the present invention are preferably provided in an isolated form, and preferably are purified to homogeneity.
In one aspect the present invention relates to a vaccine for treating or preventing pneumococcal bacterial infections which utilizes as an immunogen at least one polypeptide truncate of a pneumococcal surface-binding protein, analog, or variant having a highly conserved immunogenic alpha-helical portion (corresponding generally to a xe2x80x9cconsensusxe2x80x9d amino acid sequence as set forth in SEQ ID NO:1) with respect to different types of pneumococcal bacteria, which polypeptide does not include a choline-binding portion. Preferably, the C-terminal choline-binding portion is absent from such polypeptides. More preferred are such polypeptides wherein the HPS amino acid sequence is also absent. Even further preferred are polypeptides wherein the highly conserved immunogenic alpha-helical portion corresponding generally to a xe2x80x9cconsensusxe2x80x9d amino acid sequence as set forth in SEQ ID NO:1 also corresponds generally to the amino acid sequence as set forth in SEQ ID NO:19 (amino acids 1 to 103 of SEQ ID NO:19 are identical to amino acids 1 to 103 of SEQ ID NO:1). Also preferred as vaccines are recombinantly-produced, isolated polypeptides that are missing both an HPS portion and the choline-binding portion.
More preferred as vaccines are one or more polypeptide truncates of pneumococcal surface-binding proteins, analogs or variants including a single highly conserved alpha-helix immunogenic portion with respect to different types of pneumococci, which polypeptides do not include a C-terminal choline-binding portion. Further preferred are isolated recombinantly produced polypeptides having such structure. Also preferred are such polypeptides that do not include either a C-terminal choline-binding portion or a HPS portion.
The present invention further provides a vaccine comprising a polypeptide including an immunogenic portion that is capable of forming an alpha helix, which polypeptide includes a sequence that has at least 85% identity and preferably at least 87% identity to the amino acid sequence of SEQ ID NO:1, wherein the isolated polypeptide does not include a C-terminal choline-binding portion. Further preferred are such polypeptides that comprise a polypeptide sequence that has at least 85% identity and preferably at least 87% identity to an amino acid sequence according SEQ ID NO:19. Preferably, the sequence of the isolated polypeptide includes neither an HPS portion (SEQ ID NO:2) nor a C-terminal choline-binding portion. Further preferred are isolated recombinantly produced polypeptides having such structure. In particular, such polypeptides corresponding to alpha helical structures of different types of S. pneumoniae bacteria are contemplated. Particularly preferred are the serotypes 1-5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19F, 19A, 20, 22F, 23F and 33F of such S. pneumoniae bacteria. Examples of such serotypes of bacteria are readily available from standard ATCC catalogs.
In an additional aspect, the present invention further provides a vaccine against S. pneumoniae comprising a synthetic or recombinant polypeptide comprising a plurality of alpha-helical portions, each derived from different naturally occurring S. pneumoniae choline-binding polypeptides wherein such alpha-helical portions have at least 85% identity to the amino acid sequence of SEQ ID NO:1, and wherein the isolated polypeptide does not include a choline-binding portion. Further preferred are those wherein the amino acid sequence for the alpha-helix areas is at least 85% identical to the amino acid sequence of SEQ ID NO:19. Preferably, such synthetic polypeptide includes neither a HPS portion nor a choline-binding portion. Analogs and variants of such chain structure polypeptides wherein such alpha helical portions may be synthetic variant amino acid sequences (or may be a mixture of naturally occurring and variant sequences) are also contemplated and embraced by the present invention. In a preferred aspect, chain vaccines polypeptides having at least ten different alpha helical structures corresponding to S. pneumoniae serotypes 1-5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19F, 19A, 20, 22F, 23F and 33F are provided. Further preferred are polypeptides including at least fifteen of such alpha-helical structures, more preferred are polypeptides including at least 20 such alpha-helical structures and more preferred are polypeptides including at least one alpha-helical structure corresponding to each of the S. pneumoniae serotypes 1-5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19F, 19A, 20, 22F, 23F and 33F. Another preferred polypeptide comprises each of the alpha helical structures from the amino acid sequences of SEQ ID NOS:3-18 which correspond to SEQ ID NO:1.
In another aspect, the invention relates to passive immunity vaccines formulated from antibodies against a polypeptide including a highly conserved immunogenic portion with respect to different types of pneumococcal bacteria which portion is capable of forming an alpha-helix having the hereinbefore described identity to the amino acid sequence of SEQ ID NO:1, which polypeptide does not include a C-terminal choline-binding portion, wherein said antibodies will bind to at least one S. pneumoniae species. Preferably, if such polypeptide is a truncate of a native pneumococcal surface-binding protein both its HPS portion (where applicable) and its choline-binding portion are absent from such polypeptide. Such passive immunity vaccines can be utilized to prevent and/or treat pneumococcal infections in immunocompromised patients, patients having an immature immune system (such as young children) or patients who already have an ongoing infection. In this manner, according to a further aspect of the invention, a vaccine can be produced from a synthetic or recombinant polypeptide wherein the polypeptide includes the conserved alpha helical portions of two or more different choline binding polypeptides of S. pneumoniae. 
This invention also relates generally to the use of an isolated polypeptide having a highly conserved immunogenic portion with respect to different types of pneumococcal bacteria which portion is capable of forming an alpha-helix (corresponding generally to SEQ ID NO:1 or to SEQ ID NO:19) wherein the isolated polypeptide does not include a choline-binding portion, to raise antibodies in non-human mammalian species useful, for example, as diagnostic reagents and vaccines.
In yet another aspect, the present invention relates to the production of a polypeptide including a highly conserved immunogenic portion with respect to different types of pneumococcal bacteria which portion is capable of forming an alpha-helix whose sequence corresponds generally to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:19, wherein the isolated polypeptide does not include a choline-binding portion. Preferably, such recombinant production is of a truncated native pneumococcal surface-binding polypeptide wherein both the HPS portion (where applicable) and the choline-binding portion are absent.
In still another aspect, the present invention provides an isolated choline-binding polypeptide, wherein the non-choline binding region of such polypeptide has at least 90% identity to the corresponding amino acid sequence portion of a naturally occurring pneumococcal surface-binding protein which is a member selected from the group consisting of SEQ ID NOS:3-18. The invention relates to fragments of such polypeptides which include at least the conserved alpha-helical portion corresponding generally to SEQ ID NO:1, and which has at least 85% identity thereto, wherein the isolated polypeptide preferably is free of a choline binding region.
In another aspect the present invention provides an isolated polypeptide comprising an amino acid sequence which has at least 90% identity to one of the amino acid sequences selected from the group consisting of SEQ ID NO:3-18. Preferably, such isolated polypeptide comprises an amino acid sequence which has at least 95% identity, and more preferably 97% identity, to one of the amino acid sequences selected from the group consisting of SEQ ID NO:3-18. The invention further relates to fragments of such polypeptides.
In a yet further aspect, the present invention provides a S. pneumoniae CBP polypeptide encoded by a polynucleotide that will hybridize under highly stringent conditions to the complement of a polynucleotide encoding a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOS:1 and 3-8. Particularly preferred are polypeptides comprising an amino acid sequence segment that is at least 90% identical to the amino acid sequence of SEQ ID NO:1. Further preferred are such polypeptides comprising a contiguous amino acid sequence that has at least 95% identity with respect to the amino acid sequence of SEQ ID NO:1. And, even more preferred are polypeptides comprising an amino acid sequence that has at least 97% identity with respect to the amino acid sequence of SEQ ID NO:1.
In another aspect the present invention provides polynucleotides which encode the hereinabove described polypeptides of the invention. The polynucleotide of the present invention may be in the form of RNA or in the form of DNA, which DNA includes cDNA, genomic DNA, and synthetic DNA. The DNA may be double-stranded or single-stranded, and if single stranded may be the coding strand or non-coding (anti-sense) strand. The polynucleotides which encode polypeptides including the amino acid sequences of at least one of SEQ ID NOS:3-18 (or polypeptides that have at least 90% identity to the amino acid sequences of such polypeptides) may be one of the coding sequences shown in SEQ ID NOS:20-35 or may be of a different coding sequence which coding sequence, as a result of the redundancy or degeneracy of the genetic code, encodes the same polypeptides as the DNA of SEQ ID NOS:20-35.
The polynucleotides which encode the polypeptides of SEQ ID NOS:3-18 may include: only the coding sequence for the polypeptide; the coding sequence for the polypeptide (and optionally additional coding sequence) and non-coding sequence, such as introns or non-coding sequence 5xe2x80x2 and/or 3xe2x80x2 of the coding sequence for the polypeptide. The polypeptides encoded may comprise just a single alpha-helical portion or multiple alpha-helical portion and may independently or collectively include N-terminal sequences 5xe2x80x2 of such alpha helical areas and/or sequences corresponding to the xe2x80x9cXxe2x80x9d structures or proline rich areas (as set forth in FIG. 1, for example).
The invention further relates to a polynucleotide comprising a polynucleotide sequence that has at least 95% identity and preferably at least 97% identity to a polynucleotide encoding one of the polypeptides comprising SEQ ID NO:3-18. The invention further relates to fragments of such polynucleotides which include at least the portion of the polynucleotide encoding the polypeptide sequence corresponding to SEQ ID NO:1.
Thus, the term xe2x80x9cpolynucleotide encoding a polypeptidexe2x80x9d encompasses a polynucleotide which includes only coding sequence for the polypeptide as well as a polynucleotide which includes additional coding and/or non-coding sequence. In particular, the polypeptides may include any or all of the types of structures set forth schematically in FIG. 1.
The present invention further relates to variants of the hereinabove described polynucleotides which encode for fragments, analogs and derivatives of the polypeptides including the amino acid sequences of SEQ ID NOS:3-18. The variants of the polynucleotides may be a naturally occurring allelic variant of the polynucleotides or a non-naturally occurring variant of the polynucleotides. Complements to such coding polynucleotides may be utilized to isolate polynucleotides encoding the same or similar polypeptides. In particular, such procedures are useful to obtain alpha helical coding segments from different serotypes of S. pneumoniae, which is especially useful in the production of xe2x80x9cchainxe2x80x9d polypeptide vaccines containing multiple alpha helical segments.
Thus, the present invention includes polynucleotides encoding polypeptides including the same polypeptides as shown in the Sequence Listing as SEQ ID NOS:3-18 as well as variants of such polynucleotides which variants encode for a fragment, derivative or analog of the polypeptides of SEQ ID NOS:3-18. Such nucleotide variants include deletion variants, substitution variants and addition or insertion variants.
As hereinabove indicated, the polynucleotides may have a coding sequence which is a naturally occurring allelic variant of the coding sequence shown in the Sequence Listing as SEQ ID NOS:20-35. As known in the art, an allelic variant is an alternate form of a polynucleotide sequence which may have a substitution, deletion or addition of one or more nucleotides, which does not substantially alter the function of the encoded polypeptide.
The polynucleotides of the present invention may also have the coding sequence fused in frame to a marker sequence which allows for purification of the polypeptides of the present invention. The marker sequence may be, for example, a hexa-histidine tag supplied by a pQE-9 vector to provide for purification of the mature polypeptides fused to the marker in the case of a bacterial host, or, for example, the marker sequence may be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells, is used. The HA tag corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson, I., et al., Cell, 37:767 (1984)).
The present invention further relates to polynucleotides (hybridization target sequences) which hybridize to the complements of the hereinabove-described sequences if there is at least 70% and preferably 80% identity between the target sequence and the complement of the sequence to which the target sequence hybridizes, preferably at least 85% identity. More preferred are such sequences having at least 90% identity, preferably at least 95% and more preferably at least 97% identity between the target sequence and the sequence of complement of the polynucleotide to which it hybridizes. The invention further relates to the complements to both the target sequence and to the polynucleotide sequence that encodes an amino acid sequence selected from the group consisting of SEQ ID NOS:3 to 18. The present invention particularly relates to polynucleotides which hybridize under stringent conditions to the complements of the hereinabove-described polynucleotides as well as to those complements. As herein used, the term xe2x80x9cstringent conditionsxe2x80x9d means hybridization will occur with the complement of a polynucleotide and a corresponding sequence only if there is at least 95% and preferably at least 97% identity between the target sequence and the sequence of complement of the polynucleotide to which it hybridizes. The polynucleotides which hybridize to the complements of the hereinabove described polynucleotides in a preferred embodiment encode polypeptides which retain an immunogenic portion that will cross-react with an antibody to at least one of the polypeptides having a sequence according to SEQ ID NOS:3-18, or to a polypeptide that includes an amino acid sequence which has at least 85% identity to that of SEQ ID NO:1.
In a still further aspect, the present invention provides for the production of such polypeptides and vaccines as set forth above having a histidine label (or other suitable label) such that the full-length proteins, truncates, analogs or variant discussed above can be isolated due to their label.
In another aspect the present invention relates to a method of prophylaxis and/or treatment of diseases that are mediated by pneumococcal bacteria that have surface-binding CBP proteins. In particular, the invention relates to a method for the prophylaxis and/or treatment of infectious diseases that are mediated by S. pneumoniae that have a CBP surface-binding protein that forms an alpha helix (comprising a sequence that has at least an 85% identity to the amino acid sequence of SEQ ID NO:1). In a still further preferred aspect, the invention relates to a method for the prophylaxis and/or treatment of such infections in humans.
In still another aspect the present invention relates to a method of using one or more antibodies (monoclonal, polyclonal or sera) to the polypeptides of the invention as described above for the prophylaxis and/or treatment of diseases that are mediated by pneumococcal bacteria that have CBP surface-binding proteins. In particular, the invention relates to a method for the prophylaxis and/or treatment of infectious diseases that are mediated by S. pneumoniae CBP proteins which include an alpha helical portion having the hereinbefore described identity to the consensus sequence of SEQ ID NO:1. In a still further preferred aspect, the invention relates to a method for the prophylaxis and/or treatment of otitis media, nasopharyngeal, bronchial infections, and the like in humans by utilizing antibodies to the alpha-helix containing immunogenic polypeptides of the invention as described above.